This invention relates generally to analysis of phosphorylated compounds, and more specifically to detecting phosphorylation of biological molecules.
The cells of all organisms are capable of evaluating changes in their environment and appropriately altering function for an appropriate response. The responses are mediated by signal transduction systems that convert external stimuli into internal biochemical signals. These signal transduction systems share several characteristics across a wide variety of organisms whether single cell organisms such as bacteria or multicellular organisms such as animals and plants. Typically, a specific cellular receptor changes from one state to another due to a particular environmental stimulus and this change in state initiates a series of biochemical reactions that lead to one or more functional responses of the cell to the stimulus. For example, the presence of a particular nutrient, toxin or hormone can be detected by a cell leading to a change in cellular function allowing uptake and metabolism of the nutrient, sequestration and breakdown of the toxin or changes in growth and development, respectively.
One of the most widely used biochemical reactions of the signal transduction pathways of a wide variety of organisms is the transfer of a phosphate residue onto a biological target molecule in such a way that the function of the target molecule is changed. The addition of phosphates is mediated by kinases. Typically, a series of protein kinases act to sequentially activate each other by phosphorylation until ultimately the pathway reaches an enzyme that, when phosphorylated, changes its function to adapt the cell to the initial stimulus that activated the signal transduction pathway.
Signal transduction pathways that utilize phosphorylation play critical roles in a multitude of cellular functions including cell homeostasis, differentiation, development and growth. Improper function or response of signal transduction pathways have been found to play important roles in a variety of diseases and conditions including, for example, cancer, allergic responses, autoimmune diseases and degenerative diseases. In addition, the efficacy of many therapeutic drugs is due to their ability to alter particular signal transduction pathways.
The ability to monitor the states of phosphorylation for specific biochemical molecules can provide an understanding of many fundamental biological processes, for example, in a research setting. This can also be beneficial in a clinical setting for the diagnosis of various diseases or conditions, or for the prognosis of individuals being treated for a particular disease or condition. Furthermore, the ability to monitor phosphorylation can be valuable for the design or identification of therapeutic drugs that target specific kinases or signal transduction pathways.
Currently, several technologies are available for analyzing and detecting phosphorylation of biological molecules. One of the most widely used methods is to treat a test sample with ATP containing radioactive γ-phosphate followed by evaluating incorporation of radioactivity into a target component of interest. In many cases, the use of radioactivity is undesirable due to safety concerns, the cost of preparing and handling radioactive material and the environmental impacts of radioactivity disposal. Radioactivity can be avoided by using a phospho-specific antibody to detect a phosphorylated target component. However, the ability of these antibodies to detect a phosphate residue is dependent upon the chemical composition of the component to which the phosphate residue is attached. In many cases a new antibody must be generated for each different target to be detected. This can lead to difficulties when dealing with biological targets for which the exact composition is unknown. Even when targets of known composition are used, the need to generate and evaluate different antibodies for each target can be both time consuming and expensive.
Thus, there exists a need for a method of detecting phosphorylation of biological targets that is safe, effective and widely applicable to targets having any of a variety of different chemical compositions. The present invention satisfies this need and provides other advantages as well.